Oligosaccharide production in yeast cells expressing an ABC transporter protein

Abstract

Provided herein are genetically modified yeast cells capable of producing one or more human milk oligosaccharides (HMOs) and methods of making such cells. The yeast cells are engineered to comprise a heterologous nucleic acid encoding a transporter protein and one or more heterologous nucleic acids that encode enzymes of a HMO biosynthetic pathway. Also provided are fermentation compositions including the disclosed genetically modified yeast cells, and related methods of producing and recovering HMOs generated by the yeast cells.

What is claimed is: 
     
       1. A yeast cell genetically modified to produce one or more human milk oligosaccharides, wherein the yeast cell comprises (i) a heterologous nucleic acid encoding a human milk oligosaccharide (HMO) ABC transporter polypeptide; and (ii) one or more heterologous nucleic acids that each independently encode at least one enzyme of a human milk oligosaccharide biosynthetic pathway. 
     
     
       2. The yeast cell of  claim 1 , wherein the ABC transporter exports the human milk oligosaccharide 2′-fucosyllactose. 
     
     
       3. The yeast cell of  claim 1 , wherein the ABC transporter has at least 95% identity to any one of SEQ ID NOS: 1-27. 
     
     
       4. The yeast cell of  claim 3 , wherein the ABC transporter has at least 95% identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. 
     
     
       5. The yeast cell of  claim 1 , wherein the one or more human milk oligosaccharides comprise 2′-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, 3′-sialyllactose, 6′-sialyllactose, and/or difucosyllactose. 
     
     
       6. The yeast cell of  claim 5 , wherein the enzymes encoded by the one or more heterologous nucleic acids that independently encode at least one enzyme of the human milk oligosaccharide biosynthetic pathway comprise one or more of a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, an α-1,2-fucosyltransferase, a fucosidase, β-1,3-N-acetylglucosaminyltransferase, a β-1,3-galactosyltransferase, a UDP-N-acetylglucosamine diphosphorylase, a β-1,4-galactosyltransferase, a CMP-Neu5Ac synthetase, a sialic acid synthase, a UDP-N-acetylglucosamine 2-epimerase, a CMP-N-acetylneuraminate-β-galactosamide-α-2,3-sialyltransferase, and an α-1,3-fucosyltransferase. 
     
     
       7. The yeast cell of  claim 1 , wherein the enzymes encoded by the one or more heterologous nucleic acids further comprise a protein that transports lactose into the yeast cell. 
     
     
       8. A method of producing one or more human milk oligosaccharides, the method comprising culturing a population of genetically modified yeast cells of  claim 1  in a culture medium under conditions suitable for the yeast cells to produce the one or more human milk oligosaccharides. 
     
     
       9. A fermentation composition comprising:
 a population of genetically modified yeast cells comprising the yeast cell of  claim 1 ; and a culture medium comprising one or more human milk oligosaccharides produced from the yeast cells.